23582

CCNDBP1

DIP1|GCIP|HHM;cyclin D-type binding-protein 1;CCNDBP1;cyclin D-type binding-protein 1

D-type cyclin-interacting protein 1|cyclin-D1-binding protein 1|grap2 and cyclin-D-interacting protein|human homolog of Maid

15q14-q15

protein-coding

Transcription factors with helix-loop-helix (HLH) motif play critical roles in controlling the expression of genes involved in lineage commitment, cell fate determination, proliferation, and tumorigenesis. To examine whether the newly identified HLH protein GCIP/CCNDBP1 modulates cell fate determination and plays a role in hepatocyte growth, proliferation, and hepatocarcinogenesis, we generated transgenic mice with human GCIP gene driven by a liver-specific albumin promoter. We demonstrated that in GCIP transgenic mice, the overall liver growth and regeneration occurred normally after liver injury induced by carbon tetrachloride (CCl4). In the diethylnitrosamine (DEN)-induced mouse hepatocarcinogenesis, we demonstrated that overexpression of GCIP in mouse liver suppressed DEN-induced hepatocarcinogenesis at an early stage of tumor development. The number of hepatic adenomas at 24 weeks was significantly lower or not detected in GCIP transgenic male mice compared to the control mice under the same treatment. Although GCIP has little inhibition on the number of hepatic tumors at later stages (40 weeks), hepatocellular tumors in GCIP transgenic mice are smaller and well-differentiated compared to the poorly differentiated tumors in wild-type mice. Furthermore, we demonstrate that GCIP functions as a transcriptional suppressor, regulates the expression of cyclin D1, and inhibits anchorage-independent cell growth and colony formation in HepG2 cells, suggesting a significant role of GCIP in tumor initiation and development.

The ribosomal acidic P0 protein, an essential component of the eukaryotic ribosomal stalk, was found to interact with the helix-loop-helix protein human Grap2 and cyclin D interacting protein (GCIP)/D-type cyclin-interacting protein 1/human homolog of MAID protein. Using in vivo and in vitro binding assays, we show that P0 can interact with the N and C termini of GCIP via its N-terminal 39-114 amino-acid residues. Although the P0-GCIP complex was detected mainly in cytoplasmic fraction, polysome profile analysis indicated that the P0-GCIP complex did not coelute with either polysomes or 60S ribosomes, suggesting that GCIP associates with the free form of P0 in the cytoplasm. Transfection of GCIP into MCF-7 cells resulted in decreased levels of pRb phosphorylation. Cotransfection of P0 with GCIP, however, resulted in GCIP-mediated reduction of pRb phosphorylation level which was repressed by P0. Furthermore, overexpression of P0 in breast cancer and hepatocellular cancer cell lines promoted cell growth and colony formation compared to control transfectants. Overexpression of P0 also increased cyclin D1 expression and phosphorylation of pRb at Ser780. Interestingly, P0 mRNA was overexpressed in 12 of 20 pairs of breast cancer/ normal breast specimens (60%). Together, these data indicate that P0 overexpression may cause tumorigenesis in breast and liver tissues at least in part by inhibiting GCIP-mediated tumor suppression.

Ras associated with diabetes (Rad) is a Ras-related GTPase that promotes cell growth by accelerating cell cycle transitions. Rad knockdown induced cell cycle arrest and premature senescence without additional cellular stress in multiple cancer cell lines, indicating that Rad expression might be critical for the cell cycle in these cells. To investigate the precise function of Rad in this process, we used human Rad as bait in a yeast two-hybrid screening system and sought Rad-interacting proteins. We identified the Grap2 and cyclin D interacting protein (GCIP)/DIP1/CCNDBP1/HHM, a cell cycle-inhibitory molecule, as a binding partner of Rad. Further analyses revealed that Rad binds directly to GCIP in vitro and coimmunoprecipitates with GCIP from cell lysates. Rad translocates GCIP from the nucleus to the cytoplasm, thereby inhibiting the tumor suppressor activity of GCIP, which occurs in the nucleus. Furthermore, in the presence of Rad, GCIP loses its ability to reduce retinoblastoma phosphorylation and inhibit cyclin D1 activity. The function of Rad in transformation is also evidenced by increased telomerase activity and colony formation according to Rad expression level. In vivo tumorigenesis analyses revealed that tumors derived from Rad knockdown cells were significantly smaller than those from control cells (P = 0.0131) and the preestablished tumors are reduced in size after the injection of siRad (P = 0.0064). Therefore, we propose for the first time that Rad may promote carcinogenesis at least in part by inhibiting GCIP-mediated tumor suppression.